PROJECT SUMMARY Animal cell proliferation is regulated by cell-extrinsic cues including nutrients, cell adhesion, and growth factors produced by other cells. Textbook models hold that these inputs trigger phosphorylation cascades that culminate in the transcription of genes encoding G1 Cyclins, which in turn promote DNA replication and cell division. But studies in yeast, Drosophila, mice and human cells suggest that this view may be incomplete, or fundamentally incorrect. We posit a different mechanism in which growth-dependent translation of limiting, unstable cell cycle regulatory proteins - ?growth sensors? - determines whether and how fast cells proliferate. Unlike prevailing models this mechanism automatically couples rates of protein synthesis and cell growth to DNA replication and cell division. We seek to validate this model in Drosophila and human cells, and determine its specific mechanisms. We have two specific aims. Aim 1 builds on our discovery that Drosophila E2F1, a master regulator of cell cycle gene transcription, acts as a translationally regulated growth sensor in several fly cell types. We apply assays in cultured S2 cells and four cell types in vivo to determine how E2F1 translation is regulated by specific sequences in its UTRs. In two of these cell types (salivary and wing cells), E2F1 accumulation and cell cycle progression are driven by Insulin/Pi3K/Tor signaling. In two others (intestinal stem cells, enterocytes), EGFR/Ras/Erk signaling is the principal growth stimulus upstream of E2F1. Thus these tests may distinguish cell type- or pathway-specific modes of cell cycle control. After mapping the critical E2F1 UTR sequences in each cell type, we address their linkage to growth signaling using a whole-genome RNAi screen for trans-acting regulators. Aim 2 tests whether the growth sensor model applies in normal human epithelial (RPE-1) cells. We use ribosome profiling to identify cell cycle genes that are translationally regulated during G0/G1/S transitions by serum, ERK, mTOR, and adhesion signaling. We follow up with functional tests of candidates to identify genes that regulate quiescence/proliferation decisions and/or proliferation rates via translational control. Once identified, bona fide human growth sensors will be analyzed by the same strategy as applied to fly E2F1: growth-sensing UTR sequences will be mapped and used to build reporters, which are employed in a CRISPR screen for trans-acting factors that link the gene's translation to growth signaling. Overall this project promises to advance our understanding of how normal and oncogenic signaling pathways including PI3K/mTOR and EGFR/RAS/ERK regulate cell proliferation during normal development, regeneration, and in disease. Revising the prevailing paradigm for how growth signaling regulates the cell cycle could change what's taught in university classrooms, provide a new basis for understanding how metabolism impacts growth, and present new strategies and gene targets for the diagnosis, treatment, and prevention of diseases of dysregulated cell proliferation, most obviously cancer.